Heat-stable enzymes are essential tools of molecular biology that have proven invaluable in DNA cloning, sequencing, and random mutagenesis. The most often used heat-stable polymerases are the DNA polymerases utilized in polymerase chain reactions (PCR) reactions. These are often coupled with reverse transcriptases in RT-PCR, when an RNA molecule is used as a template to form a complementary DNA (cDNA) molecule, and that cDNA sequence is amplified by polymerase chain reaction (PCR). cDNA synthesis is most often done using reverse transcriptase enzymes of viral origin, with the reactions being performed at temperatures below about 50° C., which is optimal for these enzymes. There are, however, distinct advantages to synthesizing cDNA at temperatures above 50° C., since higher temperatures melt the secondary structures that can form in the RNA template and block further processivity of the transcriptase enzyme. A reverse transcriptase that remains stable and active at higher temperatures is especially useful for cDNA synthesis in combination reverse transcription/polymerase chain reaction (RT-PCR) reactions, as well as in other applications. Since higher temperatures can eliminate the secondary structures that may form in the RNA template, the length of the cDNA product can be extended. Higher temperatures also reduce the amount of non-specific annealing of PCR primers during cDNA synthesis, increasing specificity and amplification of cDNA. Higher temperatures can also melt the 3′ end of a mismatched primer, inhibiting further synthesis from the primer and limiting incorporation of mismatched bases in the cDNA product.
Retroviral reverse transcriptases generally have three potential enzymatic activities associated with them: an RNA-directed DNA polymerase, a DNA-directed DNA polymerase, and an RNAse H activity. Therefore, when retroviral RTs are used to copy RNA or DNA, an RNAse inhibitor must often be included in the reaction to minimize RNAse effects. Unfortunately, this can also inhibit the action of other enzymes. Furthermore, retroviral enzymes are typically most effective at temperatures at or below 50° C.
To produce cDNA at higher temperatures, a DNA-dependent, DNA polymerase Tth pol, from the thermophilic bacterium Thermus thermophilus, has been used. Although it is not technically classified as a reverse transcriptase, it will reverse transcribe RNA at high temperatures in an RT-PCR reaction. Manganese chloride (MnCl2) must be added to the reaction to boost efficiency, but this also reduces the fidelity of cDNA synthesis so that an added step is necessary to remove it prior to PCR amplification.
An RNase H-deficient Avian Myeloblastosis Virus reverse transcriptase (AMV-RT) has been used in RT-PCR reactions, the RNase H-deficient enzyme being more thermostable than the native enzyme. This RT does not degrade the RNA template, increasing the amount of full-length cDNA product that can be produced. cDNA synthesis with this enzyme is generally performed at 50° C., but larger amounts of the enzyme and substrate dNTPs are required for cDNA synthesis at higher temperatures, and synthesis from long RNA templates is often truncated, even at the higher temperature. AMV-RT also comprises two polypeptide chains (α and β), making it more difficult and expensive to produce as a recombinant product. When expressed in E. coli, for example, the end product is not appropriately modified to provide a fully active enzyme. An RT from Moloney Murine Leukemia Virus (MMLV) is also commercially available for use in reverse transcription reactions. Invitrogen's (Carlsbad, Calif.) SuperscriptII® RT is a point mutant of M-MLV-RT. According to the product literature, SuperscriptII® RT can be used at temperatures up to 50° C., and native M-MLV-RT can be used at temperatures up to 42° C.
Although there are currently enzymes which can be used in RT-PCR and other similar types of reactions, there is still a significant need for an improved RT that remains active, accurate, and stable at high temperatures and can be used in a one-step reaction system for RT-PCR.